Improved Transgene Expression in Pluripotent Stem Cells and their Differentiated Progeny by the Use of Ubiquitous Chromatin Opening Elements (UCOE)

• Applicant: Professor Dr. Thomas Moritz
• Subject Area: Hematology, Oncology
• Term: from 2013 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 244040345

For the efficient genetic modification of pluripotent cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells and their differentiated progeny silencing of transgene represents one of the main obstacles. In a recent proof-of-principle study, our group demonstrated that ubiquitous chromatin opening elements (UCOE) and specifically a 1.5 kb genetic element derived from the human HNRPA2B1/CBX3-locus (A2UCOE) in murine models can overcome this problem by stabilizing transgene expression in non-differentiated ES/iPS-cells as well as their hematopoietic progeny. Employing defined murine and human differentiation models, we now want to investigate, whether this concept can be transferred also to human mesenchymal (hematopoietic and cardiac) as well as to alternative murine (neuronal, hepatic, and cardiac) differentiation pathways. Furthermore we intend to evaluate the transgene promoting activity of the A2UCOE during the in vivo-differentiation of ESC/iPSC in an established murine embryogenesis model applying tetraploid complementation technology. In addition to these more translational aspects we also want to explore the molecular basis underlying the transgene stabilizing effect of UCOEs. Here, we will perform structure-function studies utilizing defined deletion and substitution mutants of the A2UCOE. Furthermore we will analyse the role of specific viral integration sites and their epigenetic environment on the efficacy of the A2UCOE. To this end, a clonal analysis of a panel of ESC/iPSC clones with single semi-random integration will be combined with the study of clones harbouring vectors containing or not containing the A2UCOE at the same defined site. These clones will be generated by flip recombinase-mediated cassette exchange (FRMCE) and it is intended to study two integrations each in transcriptionally silent heterochromatin as well as near to the transcriptional start site of actively transcribed genes. Successful completion of our project thus would establish a universal approach to circumvent transgene silencing in pluripotent stem cells and thereof derived progeny, and thereby would be of immediate practical relevance for the whole field of generating transgenic cell therapy products from pluripotent cell sources. In addition, the studies suggested here are designed to enhance our knowledge about the molecular and specifically the epigenetic determinants responsible for the efficacy of UCOEs. Hence, in the long run this project also will contribute to a better understanding of the molecular and epigenetic mechanisms governing the silencing of transgenes following retroviral gene transfer.

• DFG Programme: Research Grants